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United States Patent |
6,210,874
|
Lacroix
|
April 3, 2001
|
Synthetic peptides and mixtures thereof for detecting HIV antibodies
Abstract
Cyclic peptides of the general formulae:
##STR1##
wherein x represents an amino acid sequence from position 585 to 604
(gp41-HIV-1) with at least one of a lysine at position 586 or a lysine at
both positions 585 and 586; x.sup.2 represents an amino acid sequence from
position 585 to 604 (gp41-HIV-1); y represents an amino acid sequence from
position 612 to 629 (gp41-HIV-1); e and f represent one or more epitopes
included in the amino acid sequence extending from 586 to 629 (gp41-HIV-1)
or from 578 to 613 (gp36-HIV-2); and a and b represent the amino and
carboxy terminals, respectively, as well as substituents effective to make
the peptide more useful as an immunodiagnostic. Peptides of formula III
have one or both of e and f present. Also provided are peptides of the
general formulae:
##STR2##
wherein x.sup.1 represents an amino acid sequence from position 577 to 596
(gp36-HIV-2) with at least one of a lysine at position 578 or a lysine at
both positions 577 and 578; x.sup.3 represents an amino acid sequence from
position 577 to 596 (gp36-HIV-2); y.sup.1 represents an amino acid
sequence from position 604 to 613 (gp36-HIV-2); e and f represents one or
more epitopes included in the amino acid sequence from 578 to 613
(gp36-HIV-2) or 586 to 629 (gp41-HIV-1); and a and b are as defined above.
Peptides of formula IV have one or both of e and f present. These peptides
are useful in detecting HIV-1 and HIV-2 antibodies.
Inventors:
|
Lacroix; Martial (Brossard, CA)
|
Assignee:
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Biochem Immunosystems, Inc. (Quebec, CA)
|
Appl. No.:
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432520 |
Filed:
|
May 1, 1995 |
Current U.S. Class: |
435/5; 530/317; 530/324 |
Intern'l Class: |
C12Q 001/70 |
Field of Search: |
530/324,317
514/12
435/5
|
References Cited
U.S. Patent Documents
4629783 | Dec., 1986 | Cosand | 435/5.
|
4735896 | Apr., 1988 | Wang | 435/5.
|
4772547 | Sep., 1988 | Heimer | 435/5.
|
4812556 | Mar., 1989 | Vahlne | 530/324.
|
4879212 | Nov., 1989 | Wang | 435/5.
|
4957737 | Sep., 1990 | Heimer | 424/88.
|
5001049 | Mar., 1991 | Klein | 435/5.
|
5241047 | Aug., 1993 | Lacroix | 530/324.
|
5260189 | Nov., 1993 | Formoso et al. | 435/5.
|
Foreign Patent Documents |
0219106 | Apr., 1987 | EP | 435/5.
|
0231914 | Aug., 1987 | EP | 435/5.
|
0233045 | Aug., 1987 | EP | 435/5.
|
0247557 | Dec., 1987 | EP | 435/5.
|
2593190 | Jul., 1987 | FR.
| |
2593189 | Jul., 1987 | FR.
| |
2594229 | Aug., 1987 | FR.
| |
2593922 | Aug., 1987 | FR.
| |
2596063 | Sep., 1987 | FR.
| |
2597500 | Oct., 1987 | FR.
| |
2610632 | Apr., 1988 | FR.
| |
2614025 | Oct., 1988 | FR.
| |
87/04185 | Apr., 1989 | SE.
| |
WO/86/06414 | Nov., 1986 | WO | 435/5.
|
87/04459 | Jul., 1987 | WO.
| |
WO/87/06005 | Oct., 1987 | WO | 435/5.
|
89/03844 | May., 1989 | WO.
| |
Other References
Allizon et al. "Genetic variability of the IAFS Virus: Nucleotide Sequence
. . . " Cell, 46 (Jul. 1986), pp. 63-74.
Bologresi, D. Tibtech, vol. 8 (1990), pp. 40-45. Dayhoff. Atlas of Protein
Sequence & Structure, vol. 5, (1972), pp. 89-99.
Gallaher. "Detection of a fusion peptide sequence in the transmembrane . .
. ", Cell, 50 (1987), pp. 327-328.
Gnann et al. "Fine mapping of an immunodominant domain in the . . . " J. of
Virology, 61 (8) (Aug. 1987), pp. 2639-2641.
Gnann et al. "Diagnosis of AIDS by using a 12-amino acid . . . " J. of Inf.
Dis., vol. 156, No. 2 (Aug. 1987), pp. 261-267.
Gnann et al. "Synthetic peptide immunoassay distinguishes HIV type . . . ",
Science, vol. 237 (Sep. 1989), pp. 1346-1349.
Ratner et al. "Complete nucleotide sequence of the AIDS virus HTLV-III",
Nature, vol. 313, (Jan. 1985), pp. 275-283.
Wang et al. "Detection of antibodies of human T-lymphotropic . . . " Proc.
Natl. Acad. Sci. USA, vol. 83 (Aug. 1986), pp. 6159-6163.
|
Primary Examiner: Budens; Robert D.
Attorney, Agent or Firm: Nixon & Vanderhye
Parent Case Text
This is a continuation of application Ser. No. 08/077,085, filed Jun. 16,
1993, now abandoned, which is a division of application Ser. No.
07/549,964, filed Jul. 9, 1990, now U.S. Pat. No. 5,241,047; which is a
CIP of application Ser. No. 07/281,205, filed Dec. 8, 1988, which is a CIP
of application Ser. No. 07/185,518, filed Apr. 22, 1988, now abandoned;
which is a CIP of application Ser. No. 07/148,821, filed Jan. 27, 1988.
Claims
We claim:
1. A purified peptide having the formula
##STR19##
wherein:
a represents the H group which attaches to form the amino terminus or a
substituent effective as a coupling agent to make the peptide more useful
as an immunodiagnostic reagent without changing its antigenic properties;
b represents the OH group which attaches to form the carboxy terminus or a
substituent effective as a coupling agent to make the peptide more useful
as an immunodiagnostic reagent without changing its antigenic properties;
x is KILAVERYLKDQQLLGIWG;
y is TTAVPWNA; and
f is SGKLI.
2. A mixture comprising the following peptides:
##STR20##
wherein:
a represents the H group which attaches to form the amino terminus or a
substituent effective as a coupling agent to make the peptide more useful
as an immunodiagnostic reagent without changing its antigenic properties;
and
b represents the OH group which attaches to form the carboxy terminus or a
substituent effective as a coupling agent to make the peptide more useful
as an immunodiagnostic reagent without changing its antigenic properties.
3. A method for detecting the presence of antibodies to HIV-1, said method
comprising contacting an analyte suspected of containing said antibodies
with the peptide of claim 1 in a manner and for a time sufficient to allow
binding of said antibodies to said peptide, and detecting binding of said
antibodies to said peptide.
Description
FIELD OF THE INVENTION
The present invention relates to novel cyclic peptides and combinations
thereof alone and with linear and cyclic peptides for detecting HIV
antibodies.
BACKGROUND OF THE INVENTION
Acquired Immune Deficiency Syndrome (AIDS), AIDS related complex (ARC) and
pre-AIDS are thought to be caused by a retrovirus, the Human
Immunodeficiency Virus (HIV). The first AIDS related virus, HIV-1 (also
known as HTLV-III, LAV-1 and ARV) has been well characterized. Another
pathogenic human retrovirus named HIV-2 (formerly LAV-2) has now been
isolated from West African patients with AIDS. See, e.g., WO 87/04459.
HIV-2 has recently been shown (Guyader et al. Nature 326 662-669, 1987) to
share a number of conserved sequences with HIV-1 and the Simian
Immunodeficiency Viruses (SIV).
Even though other numbering systems are used in the art, for ease of
understanding and comparison we have adopted herein the amino acid
numbering system of Ratner et al., Nature, 313, 277-284, 1985 for the
HIV-1 proteins and that of Guyader et al., Nature 326, 662-669 (1987) for
the HIV-2 proteins. The amino acids in the peptides of this invention are
designated by the single letter code as follows: ala=A, arg=R, asn=N,
asp=D, cys=C, gln=Q, glu=E, gly=G, his=H, ile=I, leu=L, lys=K, met=M,
phe=F, pro=P, ser=S, thr=T, trp=W, tyr=Y and val=V.
The initial immunodiagnostic tests for the detection of antibodies in the
serum of patients infected with HIV-1 utilized the whole virus as antigen.
Second generation tests made use of polypeptide sequences obtained by the
recombinant DNA methodology. Cabradilla et al. Bio/Technology 4 128-133
(1985) and Chang et al. Bio/Technology 3, 905-909 (1985), for example,
refer to bacterially synthesized viral protein fragments of 82 and 102
amino acid residues, respectively. EPA 202314 and 114243 refer to
recombinant polypeptides spanning regions of gp41 and gp120 that are
immunoreactive alone or in mixtures. Shoeman et al., Anal. Biochem. 161,
370-379 (1987) refers to several polypeptides from gp41 that are
immunoreactive with antibodies present in sera from patients infected with
HIV-1. None of the above assay procedures is, however, totally acceptable.
Their lack of sensitivity is a critical shortcoming. It may permit blood
containing virus to escape detection and thereby potentially result in the
infection of blood product receivers and continued infectivity by
undiagnosed AIDS carriers. Their lack of specificity (false positives) is
also a problem--healthy individuals are told they may have AIDS. Such
false positive may be caused by impurities. They may also be caused by
shared epitopes with viruses unrelated to AIDS present in these antigen
preparations. In this regard, Gallaher, Cell 50 327-328, 1987 has reported
that a region of gp41 of HIV-1 shares a sequence of five adjacent amino
acid residues with the respiratory syncytial virus and of four equally
distributed amino acids of the measles virus F1 glycoprotein. Thus, even
highly purified recombinant polypeptides containing this region, or any
other common regions yet to be discovered, could potentially be
responsible for false positives and the attendant unacceptable
specificity. Finally, these prior art assays do not permit detection of
very low levels of HIV antibodies. This disadvantages the assays in terms
of their ability to detect AIDS infections at a very early stage, thereby
delaying the start of treatment and permitting the possible spread of
infection by blood samples and other body fluids before effective
detection of AIDS infection.
In an attempt to solve these problems, diagnostic means and methods
employing shorter HIV antigens are now being developed. Empirical methods
to identify peptide sequences corresponding to unique and highly conserved
epitopes of the HIV viruses are also now available. These methods are, for
example, capable of assisting in the selection of short amino acid
sequences which are more likely to be exposed on the surface of the native
protein and thus useful as assay tools (for a review see Hopp and Woods,
J. Immunol. Met. 88, 1-18, 1986). Although somewhat useful, these methods
are no more than indicative. Nonetheless, they have been applied to
identify epitopes present on the surface of viruses responsible for AIDS.
For example, U.S. Pat. No. 4,629,783, International Patent Appl.
PCT/US86/00831 and EPA 303224 refer to various synthetic peptides from the
p18, p25, gp41 and gp120 proteins. These peptides are advantaged by the
relative ease and lower cost with which they can be prepared and more
importantly because of the reduced risk of obtaining false positives with
them due to impurities or the presence of shared epitopes with viral
proteins not related to AIDS.
While these smaller peptides are advantaged in terms of specificity over
the earlier recombinant polypeptide and whole virus approaches to the
diagnosis of AIDS infections, they have been less than satisfactory in
terms of overall sensitivity, perhaps because the synthesized epitope is
not able to assume and maintain a conformation that is recognized by the
AIDS antibodies. Although the number of serum samples tested in each of
these cases is very limited, specificity (few if any false positives) was
found to be very high (95%-100%) with the small synthetic peptides but the
overall sensitivity varied between 80% and 100%. In fact, in the only
example where 100% sensitivity was attained only ten samples were tested.
For example, Smith et al., J. Clin. Microbiol. 25 1498-1504, 1987 refers
to two overlapping peptides, E32 and E34, that are highly immunoreactive.
No false positives, out of 240 seronegative specimens, were obtained but
the peptides missed three seropositive samples out of 322 (sensitivity of
99.1%). Wang et al. (Proc. Natl. Acad. Sci 83, 6159-6163, 1986) refers to
a series of overlapping peptides (including amino acid residues of Smith's
E32 and E34 peptides) among which one 21-mer peptide showed 100%
specificity and 98% sensitivity (out of 228 seropositive samples taken
from patients with AIDS, 224 were found positive with this peptide). And
U.S. patent application 120,027, filed Nov. 13, 1987 refers to a short
synthetic peptide spanning residues 606 to 620 (SGKLICTTAVPWNAS) of gp41
(HIV-1). This peptide is said to be immunoreactive with antibodies of
patients infected by the AIDS viruses. The specificity was also excellent
(63/63) but 6 seropositive specimens out of 57 confirmed positive could
not be detected (sensitivity of 89%).
Gnann et al. (J. Virol. 61, 2639-2641, 1987 and J. Infec. Dis 156, 261-267,
1987) also refer to a series of overlapping peptides from a suspected
immunodominant region of gp41 (HIV-1). Gnann et al. concluded that cys-605
was essential for the immunoreactivity of that segment of the gp41-(HIV-1)
protein. They reported that a peptide having the sequence SGKLIC (606-611)
was not immunoreactive with any of the 22 HIV-1 positive sera tested,
while the addition of the cysteine residue to the N-terminus restored some
immunoreactivity, 21 of 44 sera reacted with the 7-mer peptide (48%
sensitivity).
Gnann et al. (J. Virol) also speculated that the cysteine residues at
positions 605 and 611 of gp41 (HIV-1) might play a role in the antigenic
conformation of this region perhaps by the formation of a cyclic structure
via disulfide bonding. However, Gnann et al. never demonstrated that they
did have a synthetic peptide wherein the two cysteine groups were linked
by disulfide bonds.
Although Gnann et. al refers to peptides which are useful in identifying
HIV-1 antibodies, even its peptides lack 100% sensitivity. For example,
Gnann et al. (J. Virol. 61, 2639-2641, (1987)) report that while their
600-611 amino acid sequence detected 22 out of 22 positive sera, they also
reported that similar tests carried out at the Centers for Disease
Control, Atlanta, Ga. with the same 12-amino acid sequence (600-611)
missed 1 out of 79 positive sera. And Gnann et al. in J. Infect. Dis. 156,
261-267, 1987 reported that the same 12-amino acid sequence was reactive
with 131 out of 132 HIV-1 infected patients from the United States.
Gnann et al. Science 237, 1346-1349, 1987 reports a short linear synthetic
peptide spanning residues 592 to 603 of gp42 (HIV-2) that contains two
cysteines in a region homologous to the 605-611 region of gp41(HIV-1).
This peptide reacted with 5 out of 5 sera taken from HIV-2 infected
patients.
Other peptides containing amino acids 605-611 of gp41 of (HIV-1) are also
referred to in the art. WO 86/06414 refers to peptide X(39), which is
encoded by the region from about bp 7516 through bp 7593, and peptide
XIII(79) which is encoded by the region extending from about bp 7543
through bp 7593, both containing the 7-amino acid sequence 605-611. These
peptides are reported to be linear and no formation of cyclic structures
is suggested. WO 87/06005 reports that a series of synthetic peptides
encompassing the Cys(605)-Cys(611) residues of the HIV-1 envelope
glycoprotein (gp41) undergo a series of spontaneous oxidative
transformations upon solubilization in neutral or basic aqueous buffer. It
speculates that as a result, the peptides when used in ELISAs are a random
mixture of linear monomer, cyclic monomer, linear or cyclic dimers and
linear polymers of various lengths. The application did not actualy
demonstrate the presence of cyclic components and did not characterize the
other various dimers and polymers possibly present. Moreover, it
speculates that the polymer forms are the most important components for
ELISA reactivity.
In addition to perhaps being complex mixtures of various oxidative forms of
the peptide, the prior art peptides referred to above do not permit as
early detection of AIDS infection as would be desirable. For example,
Gnann et al. (J. Virol) reports that when the HIV-1 positive sera are
diluted by a factor exceeding 500, some of these diluted sera are found to
be negative thus indicating a low sensitivity of the peptide for early HIV
detection.
These problems have been addressed by employing peptides that have been
chemically cyclized to form a disulfide bridge between the relevant
cysteines. E.g., M. Lacroix et al., Comparative Performance of Cyclic
Versus Linear Peptides In An ELISA For HIV-1 And HIV-2 Specific
Antibodies, No. 3147, June 1989 AIDS Conference, Montreal, Canada; and WO
89/03344. This invention is directed to improvements in such cyclic
peptides.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a novel series
of peptides which are particularly adapted for detecting 100% of HIV-1 and
HIV-2 antibodies and which are capable of detecting such antibodies even
when present in very low levels in sera.
More specifically, the novel peptides of the present invention are selected
from substantially pure peptides of formulae I or II:
##STR3##
wherein:
x is independently selected from one of the following amino acid sequence
analogs of the amino acid sequence of gp41-HIV-1:
KILAVERYLKDQQLLGIWG- (586-604)
KKILAVERYLKDQQLLGIWG- (585-604),
amino acid sequences corresponding thereto, which sequences are derived
from homologous regions of other HIV-1 isolates, and amino acid sequences
differing from the above as a result of conservative substitutions, such
amino acid sequences being characterized by at least one of a lysine at
position 586 or a lysine at both positions 585 and 586;
y, if present, is independently selected from the group consisting of:
##STR4##
amino acid sequences corresponding thereto, which sequences are derived
from homologous regions of other HIV-1 isolates, and amino acid sequences
differing from the above as a result of conservative substitutions;
e and f, if present, are independently selected from the group consisting
of an amino acid sequence of any one of the epitopes of the region
spanning amino acids 586 to 629 of gp41 of HIV-1 or of the region spanning
amino acid sequence 578 to 613 of gp36 of HIV-2, amino acid sequences
corresponding thereto and being derived from homologous regions of other
HIV-1 or HIV-2 isolates, amino acid sequences differing from the above as
a result of conservative substitutions, and any combination of these
epitopes;
a is an amino terminus or a substitutent effective as a coupling agent
and/or to make the peptide more useful as an immunodiagnostic reagent
without changing its antigenic properties; and
b is a carboxy terminus or a substituent effective as a coupling agent
and/or to make the peptide more useful as an immunodiagnostic reagent
without changing its antigenic properties; and
##STR5##
wherein:
x.sup.1 is independently selected from one of the following amino acid
sequence analogs of the amino acid sequence of gp36-HIV-2:
KVTAIEKYLQDQARLNSWG (578-596)
KKVTAIEKYLQDQARLNSWG (577-596),
amino acid sequences corresponding thereto, which sequences are derived
from homologous regions of other HIV-2 isolates and amino acid sequences
differing from the above as a result of conservative substitutions, such
sequences being characterized by at least one of a lysine at position 578
or a lysine at both positions 577 and 578;
y.sup.1, if present, is independently selected from the group consisting
of:
##STR6##
amino acid sequences corresponding thereto, which sequences are derived
from homologous regions of other HIV-2 isolates, and amino acid sequences
differing from the above as a result of conservative substitutes; and
e, f, a and b are as defined above.
In another embodiment, the novel peptides of this invention are selected
from substantially pure peptides of the formulae III and IV:
##STR7##
wherein x.sup.2, if present, is independently selected from the group
consisting of:
##STR8##
amino acid sequences corresponding thereto, which sequences are derived
from homologous regions of other HIV-1 isolates, and amino acid sequences
differing from the above as a result of conservative substitutions; and
y, e, f, a and b are as previously defined, one or both of e or f being
present; and
##STR9##
wherein x.sup.3, if present, is independently selected from the group
consisting of:
##STR10##
amino acid sequences corresponding thereto, which sequences are derived
from homologous regions of other HIV-2 isolates, and amino acid sequences
differing from the above as a result of conservative substitutions; and
y.sup.1, e, f, a and b are as previously defined, one or both of e or f
being present.
A particularly preferred peptide of formula I is BCH-408 which has the
following sequence:
##STR11##
This peptide incorporates at the f position 619 the amino acid sequence of
the epitope located at position 606-610 (SGKLI) of gp41 of HIV-1 and at
position 586 a lysine.
A particularly preferred peptide of formula II is BCH-417 which has the
following sequence:
##STR12##
This peptide incorporates at the f position 613 the amino acid sequence of
the epitope located at position 598-602 (AFRQV) of gp36 of HIV-2 and at
position 578 lysine.
DETAILED DESCRIPTION OF THE INVENTION
Selection of Peptides for Synthesis
The peptides of this invention were synthesized on the basis of published
amino acid sequences of HIV-1 and HIV-2. However, it should be understood
that sequences derived from the homologous regions of other HIV-1 or HIV-2
isolates can be used without departing from the scope of this invention.
Epitopes in these native sequences were chosen for use as e and f, in the
peptides of this invention using various physicochemical principles that
aid in predicting which portions of a polypeptide are most likely to be
surface oriented and therefore immunogenic. These include the
hydrophilicity plots of Hopp and Woods (Proc. Natl. Acad. Sci. 78,
3824-3828, 1981), and a similar approach by Kyte and Doolittle (J. Mol.
Biol. 157, 105-132, 1982). Also, the empirical prediction of protein
conformation (Chou and Fasman, Ann. Rev. Biochem. 47, 251-276, 1978) is a
useful guide in predicting which parts of the polypeptide are likely to be
immunogenic.
The e and f epitopes of the peptides of this invention include, but are not
limited to, WGCAF (identified by Norrby et al., AIDS Research and Human
Retroviruses, Vol. 5, No. 5, 1989); KD, SGKL, and LEDQ (identified by
Norrby et al., AIDS, Vol. 3, No. 1 (1989); LKDQ, CSGKLI, and IWG
(identified by Mathiesen et al., Immunology, 67 1-7 (1989); and
ARILAVERYLKD, and SGKLICTTAVPWNAS (identified by Dopel et al., Jol. of
Vir. Meth., 25 167-178 (1989).
It is also within the scope of this invention to modify the peptides of
this invention, in order to make them more useful as immunodiagnostic
reagents without changing their antigenic properties. Such changes
include:
addition of a cysteine residue at the amino or carboxy terminus in order to
facilitate coupling of the peptide to a carrier protein with
heterobifunctional cross-linking reagents such as
sulfosuccinimidyl-4(p-maleimidophenyl)butyrate, a preferred reagent for
effecting such linkages;
addition of certain amino acids at the amino or carboxy terminus to
facilitate linking of peptides to each other, for coupling to a support or
larger peptide or for modifying the physical or chemical properties of the
peptide. Such changes may be effected, for example, by additions of
tyrosine, glutamic acid or aspartic acid, which can be used as linkers via
an esterification reaction, and lysine which can be connected by Schiff
base or amide formation; and
derivatization by amino terminal acylation, thioglycolic acid amidation,
and carboxy terminal amidation, e.g. using ammonia, methylamine. These
modifications result in changes in net charge on the peptide and can also
facilitate covalent linking of the peptide to a solid support, a carrier
or another peptide. These modifications are not likely to result in
immunoreactivity changes to the peptide.
The peptides of this invention may also be modified by various changes such
as insertions, deletions and substitutions, either conservative or
nonconservative where such changes might provide for certain advantages in
their use. These changes include preferably the following conservative
changes: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg;
phe, tyr; ala, ser; ala, thr; ala, val; ala, pro; ala, glu; leu, gln; gly,
phe; ile, ser; and ile, met. Methionine, an amino acid which is prone to
spontaneous oxidation, can also usually be replaced by norleucine without
changing antigenicity.
It may also be convenient to add a "tail" consisting of a small number
(1-10) of hydrophobic amino acids to the peptides of this invention. Such
tails may facilitate passive adsorption of a peptide to a solid support.
This modification can be made at either the COOH or NH.sub.2 termini. The
preferred addition is phe-ala-phe-ala-phe.
In accordance with this invention, the preferred cyclic peptides of formula
I are those having
x, y, e and f defined as follows:
x: KILAVERYLKDQQLLGIWG, y: TTAVPWNAS, e and f not present (BCH-87ck);
x: KKILAVERYLKDQQLLGIWG, y: TTAVPWNAS, e and f not present (BCH-266); and
x: KILAVERYLKDQQLLGIWG, y: TTAVPWNA,
f: SGKLI and e not present (BCH-408), BCH-408 being the most preferred.
The preferred cyclic peptides of formula II are those having x.sup.1,
y.sup.1, e and f defined as follows:
x.sup.1 : KVTAIEKYLQDQARLNSWG, y.sup.1 : HTTVPWVNDS and e and f not present
(BCH-202ck);
x.sup.1 : KKVTAIEKYLQDQARLNSWG, y.sup.1 : HTTVPWVNDS and e and f not
present (BCH-265); and
x.sup.1 : KVTAIEKYLQDQARLNSWG, y.sup.1 : HTTVPWVNDS and
f: AFRQV and e not present (BCH-417), BCH-417 being the most preferred.
TABLE I provides the full amino acid sequences of these preferred peptides
(disregarding possible a and b):
TABLE 1
Peptide sequences
HIV-1:
BCH-87ck:
##STR13##
BCH-266:
##STR14##
BCH-408:
##STR15##
HIV-2:
BCH-202ck:
##STR16##
BCH-265:
##STR17##
BCH-417:
##STR18##
Preparation of Linear and Cyclic Peptides
The peptides of this invention are preferably prepared using conventional
solid phase synthesis. However, other well known methods of peptide
synthesis may also be used. The resin support is any suitable resin
conventionally employed in the art for solid phase preparation of
polypeptides, preferably p-benzyloxyalcohol polystyrene and
p-methylbenzydrylamine resin. Following the coupling of the first
protected amino acid to the resin support, the amino protecting group is
removed by standard methods conventionally employed in the art of solid
phase peptide synthesis. After removal of the amino protecting group,
remaining .alpha.-amino protected and, if necessary, side chain protected
amino acids are coupled, sequentially, in the desired order to obtain the
product. Alternatively, multiple amino acid groups may be coupled using
solution methodology prior to coupling with the resin-supported amino acid
sequence.
The selection of an appropriate coupling reagent follows established art.
For instance, suitable coupling reagents are N,N'-diisopropylcarbodiimide
or N,N'-dicyclohexylcarbodiimide (DCC) either alone or preferably in the
presence of 1-hydroxybenzotriazole. Another useful coupling procedure
makes use of preformed symmetrical anhydrides of protected amino acids.
The necessary .alpha.-amino protecting group employed for each amino acid
introduced onto the growing polypeptide chain is preferably
9-fluorenylmethyloxycarbonyl (Fmoc), although any other suitable
protecting group may be employed as long as it does not degrade under the
coupling conditions and is readily removable selectively in the presence
of any other protecting groups already present in the growing molecule.
The criteria for selecting protecting groups for the side chain amino acids
are: (a) stability of the protecting group to the various reagents under
reaction conditions selective for the removal of the .alpha.-amino
protecting group at each step of the synthesis: (b) retention of the
protecting group's strategic properties (i.e. not be split off under
coupling conditions) and (c) removability of the protecting group upon
conclusion of the polypeptide synthesis and under conditions that do not
otherwise affect the polypeptide structure.
The fully protected resin-supported peptides are cleaved from the
p-benzyloxy alcohol resin with 50 to 60 percent solution of
trifluoroacetic acid in methylene chloride for 1 to 6 hours at room
temperature in the presence of appropriate scavengers such as anisole,
thioanisole, ethyl methyl sulfide, 1,2-ethanedithiol and related reagents.
Simultaneously, most acid labile side-chain protecting groups may then be
removed. More acid resistant protecting groups are removed by HF
treatment.
Cyclic peptides of this invention are prepared by the direct oxidative
conversion of protected or unprotected SH-groups to a disulfide bond by
following techniques generally known in the art of peptide synthesis. The
preferred method involves the direct oxidation of free SH-groups with
potassium ferricyanide. Such cyclic peptides are believed to assume a more
rigid conformation which may favor binding to HIV antibodies.
Peptide Mixtures and Polymers
Within the scope of this invention are larger peptides formed by the
covalently linking of one or more peptides of this invention. Polymers
(both homo and co) of these peptides are also envisioned.
Also within the scope of the present invention are other cyclic and
mixtures of the cyclic peptides of this invention and other cyclic and
linear HIV derived peptides. These mixtures have surprisingly been found
to provide high sensitivity detection of HIV-1 and HIV-2 antibodies
present in serially diluted serum samples and in seroconversion panels
(HIV-1). Also it has been found that these mixtures provide a high level
of specificity resulting in a minimal number of false positives.
Such mixtures comprise at least one cyclic peptide of the general formulae
I or III (preferably BCH-87ck, BCH-266 or BCH-408 and more preferably
BCH-408) in combination with at least one cyclic peptide of the general
formulae II or IV (preferably BCH-202ck, BCH-265 or BCH-417 and more
preferably BCH-417).
HIV Antibody Detection
The peptides and the peptide mixtures of the present invention are useful
as diagnostic reagents for the detection of AIDS-associated antibodies in
accordance with methods well-known in the art. These include ELISA,
hemagglutination, single-dot and multi-dot methods and assays. The main
advantage of the present peptides in the determination of antibodies
against AIDS resides in their specificity and high sensitivity, and
particularly their ability to detect the presence of very low levels of
AIDS infection, when compared with known antigens used so far.
According to one method for the determination of antibodies against HIV-1
or HIV-2, the so-called "Western Blotting" analysis is used [Towbin, H.,
Staehelin, T. and Gordon, J., Proc. Nat. Acad. Sci. U.S.A. 76, 4350-4354
(1979)]. According to this technique a peptide or peptides of the present
invention is (or are) applied to nitrocellulose paper. The nitrocellulose
paper is saturated and then treated with the serum to be tested. After
washing, the nitrocellulose paper is treated with an anti-human IgG
labeled with an enzyme. The enzymatic activity is then determined by a
suitable substrate. Of course, other labels, like radioactive or
fluorescence labels, may be used.
A preferred convenient and classical technique for the determination of
antibodies against HIV-1 or HIV-2 using a peptide or a peptide mixture of
the present invention is an enzyme-linked immunosorbent assay (ELISA). In
this assay, for example, a peptide, peptide mixture or combination of the
present invention is adsorbed onto, or covalently coupled to, the wells of
a microtiter plate. The wells are then treated with the sera or analyte to
be tested. After washing, anti-human IgG or antihuman IgM labeled with
peroxidase is added to the wells. The determination of the peroxidase is
performed with a corresponding substrate, e.g., with o-phenylene diamine.
Without departing from the usefulness of the illustrative assay, the
peroxidase can be exchanged by another label, e.g., by a radioactive,
fluorescence chemiluminescence or infra-red emitting label.
In the ELISA test, it is possible to use individual peptides or a
combination thereof. The latter is preferable since it allows one to
combine the most effective peptides for detecting antibodies while at the
same time excluding those that contribute to false responses. It was
discovered during the course of these studies that some serum samples gave
correct positive results with mixtures of peptides while giving equivocal
responses with individual peptides as antigen. Thus the most preferred
test for HIV-1 and HIV-2 antibodies is achieved in accordance with this
invention using a combination of peptide antigens.
Another method for the determination of antibodies against HIV-1 or HIV-2
with the peptides or mixture of peptides of this invention is an enzyme
immunological test according to the so-called
"Double-Antigen-Sandwich-Method". This method is based on the work of
Maiolini as described in Immunological Methods 20, 25-34 (1978). According
to this method, the serum or other analyte to be tested is contacted with
a solid phase on which a peptide or mixture of peptides of the present
invention has been coated (capture layer) and with a peptide or a peptide
mixture of the present invention which is labeled with peroxidase (probe
layer). The immunological reaction can be performed in one or two steps.
If the immunological reaction is performed in two steps, then a washing
step is preferably performed between the two incubations. After the
immunological reaction or reactions, a washing step may also be performed.
Thereafter, the peroxidase is determined with a substrate, e.g., with
o-phenylene diamine. Other enzymes and chromogens, including those already
described can also be employed in this assay.
Suitable solid phases are organic and inorganic polymers, such as amylases,
dextrans, natural or modified celluloses, polyethylenes, polystyrenes,
polyacrylamides, agaroses, magnetites, porous glass powders,
polyvinyldiene fluoride (kynar) and latex, the inner wall of test vessels
e.g., test tubes, titer plates or cuvettes of glass or articifial material
as well as the surface of solid bodies, e.g., rods of glass and artificial
material, rods with terminal thickening, rods with terminal lobes or
lamallae. Spheres of glass and artificial material are especially suitable
solid phase carriers.
The peptides and mixtures of peptides of the present invention are not only
useful in the determination of antibodies against HIV-1 or HIV-2, but also
indirectly for the determination of HIV-1 or HIV-2 itself since these
peptides either free, polymerized or conjugated to an appropriate carrier
are useful in eliciting antibodies, in particular monoclonal antibodies,
against HIV-1 or HIV-2. Such antibodies can be produced by injecting a
mammalian or avian animal with a sufficient amount of a peptide or mixture
of peptides of the present invention and recovering said antibodies from
the serum of said animals. Suitable host animals for eliciting antibodies
include mammals such as rabbits, horses, goats, guinea-pigs, rats, mice,
cows, sheep, etc..
Various methods which are generally known can be employed using the
peptides of this invention or mixtures thereof in the quantitative
determination of HIV-1 or HIV-2 infection. In one such procedure known
amounts of a serum sample to be assayed, radiolabeled cyclic peptide of
the present invention or mixtures of those peptides and unlabeled peptide
or mixture of peptides of the present invention are mixed together and
allowed to stand. The antibody/antigen complex is separated from the
unbound reagents by procedures known in the art, i.e., by treatment with
ammonium sulfate, polyethylene glycol, a second antibody either in excess
or bound to an insoluble support, dextran-coated charcoal and the like.
The concentration of the labeled peptide or mixture of peptides of the
present invention is determined in either the bound or unbound phase and
the HIV-1 or HIV-2 content of the sample can then be determined by
comparing the level of labeled component observed to a standard curve in a
manner known `per se`.
Another suitable quantitative method is the
"Double-Antibody-Sandwich-Assay". According to this assay the sample to be
tested is treated with two different antibodies raised against a peptide
of this invention or mixture thereof using different animals, e.g. sheep
or rabbits. Alternatively, monoclonal antibodies may be prepared using the
well-known Koehler and Milstein technique for producing monoclonal
antibodies. In order to distinguish monoclonal antibodies which are
directed against the same antigen, but against different epitopes, the
method of Stahli et al. [J. of Immunological Methods 32, 297-304 (1980)]
can be used. It is also appropriate to use a polyclonal antiserum and a
monoclonal antibody.
One of these antibodies is labeled and the other is coated on a solid
phase. The suitable solid phases are those mentioned earlier in this
application. Suitable labels are enzymes, e.g. peroxidase, radioactive
labels or fluorescence-labels. The preferred solid phase is a plastic bead
and the preferred label is horse-radish peroxidase.
The sera sample is then incubated with the solid phase antibody and the
labeled antibody. It is possible to treat the sample first with the solid
phase antibody and after washing to treat the sample with the labeled
antibody. However, it is also possible to treat the sample first with the
solid phase antibody and after a certain time with the labeled antibody.
Preferably the sample is treated together with the solid phase and the
labeled antibody.
After the immunological reaction(s), a washing step may be performed. After
washing, the label is determined according to procedures known in the art.
In the case where peroxidase is used as the label, the determination is
performed with the substrate, e.g., with o-phenylene diamine or with
tetramethylbenzidine. The amount of the labeled component is proportional
to the amount of the antigen(s) present in the sample.
The methods and assays for the determination and quantification of HIV-1,
HIV-2 or of antibodies against HIV-1 or HIV-2 as described above can be
conducted in suitable test kits comprising, in a container, a cyclic
peptide of the present invention, peptide mixtures or a combination
thereof, or antibodies against HIV-1 or HIV-2 elicited by a cyclic peptide
or a mixture of cyclic and linear peptides of the present invention.
Panel of Sera Tested
To demonstrate the surprising sensitivity and specificity of the peptides
of this invention a panel of sera was tested with illustrative peptides.
O.D. Values were obtained at 450 nm and the blank values measured with the
sample dilution buffer were not subtracted.
Samples NEIA-2*2, BBI-1-162 to 168, 87B140, 87L139, 87V103 are all negative
for HIV antibodies. Sample LSPQ-S9-1 is an early seroconverter (HIV-1).
The series labeled CAP-113 to CAP-120 corresponds to a pool of seven HIV-1
positive plasma samples serially diluted with an HIV-negative plasma.
CAP-113 is the pool diluted 50-fold with the HIV-negative plasma; CAP-114
is diluted 100-fold; CAP-115 is diluted 200-fold; etc. Similarly, the
series labeled CAP-222 to CAP-230 corresponds to a pool of seven HIV-2
confirmed seropositive plasma samples. CAP-222 is diluted by 50 with an
HIV-negative plasma; CAP-223 by 100; etc. Before the assay is done, each
sample, including the CAP-series, is further diluted by 50 with the sample
dilution buffer. In these tests, the cut-off for seropositivity is defined
as the sum of the O.D. value for sample NEIA-2*2 plus 0.100.
Results
The cyclic peptides of the present invention were coated and tested in
accordance with the ELISA test described previously. TABLE 2 compares the
sensitivity of peptide BCH-87c (586(arginine)) to the sensitivity of
peptide BCH-87ck (586(lysine)) and peptide BCH 266
(586(lysine)--585(lysine)) at progressively higher dilutions of the
antibody in the sera samples. It was found that the substitution of a
lysine for the arginine at amino acid position 586 increased the
sensitivity in detecting HIV-1 antibodies. A further increase in
sensitivity was obtained by an additional lysine at position 585. TABLE 3
compares the activity of BCH-87c, BCH-87ck, BCH-266 and BCH-408 at
progressively higher dilutions. It is evident from TABLE 3 that peptide
BCH-408, wherein an important epitope located at position 606-610 (SGKLI)
is repeated at its c-terminus, has superior sensitivity compared to the
other peptides.
It was found with the HIV-2 peptides that substituting a lysine for the
arginine at amino acid position 578 increased the sensitivity in detecting
HIV-2 antibodies. A further increase in sensitivity was obtained by an
additional lysine at position 577. TABLE 4 compares the sensitivity of the
peptide BCH-202c (578(arginine)) to the sensitivity of peptide BCH-202ck
(578(lysine)) and peptide BCH 265 (598(lysine)--577(lysine)) at
progressively higher dilutions of the antibody in the sera samples.
Peptide cocktails were also made to detect a mixture of HIV-1 and HIV-2
antibodies. TABLE 5 illustrates the sensitivity of peptide cocktail
mixtures BCH-87c and BCH-202c (arginine) versus BCH-87ck and BCH-202ck
(lysine). The peptide cocktail which included the peptides with lysine
substituted for arginine have a higher sensitivity in detecting HIV
antibodies.
TABLE 2
HIV-1
Test 314 O.D. 450 nm
Sample ID BC-87c BCH-87ck BCH-266
Dil. Buffer 0.017 0.016 0.014
NEIA-2*2 0.045 0.050 0.069
BBI-1-162 0.010 0.015 0.028
BBI-1-169 0.095 0.090 0.138
BBI-1-172 0.012 0.019 0.030
CAP-113 >2.8 >2.8 >2.8
CAP-114 >2.8 >2.8 >2.8
CAP-115 1.935 2.593 >2.8
CAP-116 1.275 1.871 2.376
CAP-117 0.739 1.158 1.569
CAP-118 0.433 0.679 0.934
CAP-119 0.231 0.392 0.567
CAP-120 0.137 0.229 0.376
CAP-222 0.059 0.069 0.095
CAP-223 0.053 0.060 0.075
CAP-224 0.051 0.054 0.084
CAP-225 0.049 0.050 0.135
CAP-226 0.046 0.050 0.140
CAP-227 0.041 0.043 0.085
CAP-228 0.038 0.042 0.091
CAP-230 0.043 0.049 0.123
TABLE 3
HIV-1
Test 350 O.D. 450
Sample ID BCH-87c BCH-87ck BCH-266 BCH-408
Dil. Buffer 0.016 0.032 0.013 0.015
NEIA-2*2 0.027 0.034 0.023 0.035
2-87-V-103 0.020 0.015 0.020 0.014
2-87-L-139 0.158 0.409 0.147 0.025
89-D-307 0.026 0.022 0.030 0.029
CAP-10 >2.8 >2.8 >2.8 >2.8
CAP-11 >2.8 >2.8 >2.8 >2.8
CAP-12 2.432 2.735 >2.8 >2.8
CAP-13 1.488 1.676 2.021 2.319
CAP-14 0.866 1.089 1.248 1.433
CAP-15 0.449 0.643 0.680 0.835
CAP-16 0.262 0.274 0.366 0.455
CAP-17 0.141 0.191 0.198 0.244
CAP-18 0.099 0.114 0.116 0.156
CAP-19 0.068 0.053 0.056 0.102
TABLE 4
HIV-2
Test 226 O.D. 450 nm
Sample ID BC-202c BCH-202ck BCH-265
Dil. Buffer 0.015 0.014 0.015
NEIA-2*2 0.020 0.027 0.022
88I-1-162 0.018 0.033 0.059
BBI-1-169 0.018 0.028 0.076
BBI-1-172 0.019 0.024 0.050
CAP-113 1.795 >2.8 >2.8
CAP-114 1.070 2.015 2.134
CAP-115 0.611 1.195 1.330
CAP-116 0.325 0.642 0.718
CAP-117 0.174 0.365 0.410
CAP-118 0.089 0.189 0.190
CAP-119 0.057 0.082 0.117
CAP-120 0.041 0.084 0.094
CAP-222 >2.8 >2.8 >2.8
CAP-223 >2.8 >2.8 >2.8
CAP-224 2.313 >2.8 >2.8
CAP-225 1.448 2.418 2.480
CAP-226 0.758 1.436 1.674
CAP-227 0.448 0.745 0.960
CAP-228 0.233 0.409 0.363
CAP-230 0.139 0.279 0.272
TABLE 5
HIV-1
Comparative Performance of Two Peptide-Cocktails
O.D. 450 nm
Test 310 BC-87c BCH-87ck
Sample ID BC-202c BCH-202ck
Dil. Buffer 0.015 0.013
NEIA-2*2 0.034 0.062
BBI-1-162 0.044 0.034
BBI-1-163 0.032 0.042
BBI-1-164 0.052 0.077
BBI-1-165 0.038 0.059
BBI-1-166 0.045 0.081
BBI-1-167 0.020 0.064
BBI-1-168 0.031 0.041
87-B-140 0.005 0.015
87-L-139 0.025 0.068
87-V-103 0.022 0.035
CAP-113 >2.8 >2.8
CAP-114 2.634 >2.8
CAP-115 1.720 >2.8
CAP-116 0.965 2.459
CAP-117 0.548 1.685
CAP-118 0.302 1.036
CAP-119 0.158 0.609
CAP-120 0.104 0.341
CAP-222 >2.8 >2.8
CAP-223 2.470 >2.8
CAP-224 1.709 >2.8
CAP-225 1.003 2.441
CAP-226 0.554 1.733
CAP-227 0.289 1.001
CAP-228 0.169 0.611
CAP-230 0.086 0.343
LSPQ-S9-1 0.123 1.273
BBI-A-01 0.067 0.078
BBI-A-02 0.114 0.130
BBI-A-03 0.180 1.069
BBI-A-04 2.401 >2.8
BBI-A-05 >2.8 >2.8
BBI-A-06 >2.8 >2.8
BBI-A-07 >2.8 >2.8
BBI-A-08 >2.8 >2.8
BBI-A-09 >2.8 >2.8
BBI-C-20 0.019 0.050
BBI-C-21 0.017 0.048
BBI-C-22 0.030 0.121
BBI-C-24 0.210 1.748
BBI-C-25 0.372 2.452
BBI-C-26 0.374 2.353
The following illustrates the general procedures for the synthesis and
utilization of the peptides of this invention.
Procedure 1
Preparation of Resins Carrying the N.alpha.-FMOC Protected Amino Acid
Residue
The desired N.alpha.-FMOC protected amino acid residue in a mixture of
methylene chloride (CH.sub.2 Cl.sub.2) and dimethylformamide (DMF) (4:1)
was added to a suspension of p-benzyloxy alcohol resin in CH.sub.2
Cl.sub.2 :DMF (4:1) at 0.degree. C. The mixture was stirred manually for a
few seconds and then treated with N,N'-dicyclohexylcarbodiimide (DCC)
followed by a catalytic amount of 4-(dimethylamino)pyridine. The mixture
was stirred at 0.degree. C. for an additional 30 minutes and then at room
temperature overnight. The filtered resin was washed successively with
CH.sub.2 Cl.sub.2, DMF and isopropanol (3 washes each) and finally, with
CH.sub.2 Cl.sub.2. The resin was suspended in CH.sub.2 Cl.sub.2, chilled
in an ice bath and redistilled pyridine was added to the stirred
suspension Benzoyl chloride was then also added. Stirring was continued at
0.degree. C. for 30 minutes and then at room temperature for 60 minutes.
After filtration, the resin was washed successively with CH.sub.2
Cl.sub.2, DMF and isopropanol (3 washes each) and finally with petroleum
ether (twice) before being dried under high vacuum to a constant weight.
Spectrophotometric determination of substitution according to Meienhofer
et al. (Int. J. Peptide Protein Res., 13, 35, 1979) indicates the degree
of substitution on the resin.
Procedure 2
Coupling of Subsequent Amino Acids
The resin carrying the N.alpha.-FMOC protected first amino acid residue was
placed in a reaction vessel of a Biosearch 9600 Peptide Synthesizer and
treated as follows:
1) Washed with DMF (4 times for 20 sec. each)
2) Prewashed with a 30% solution of piperidine in DMF (3 min.)
3) Deprotected with a 30% solution of piperidine in DMF (7 min.)
4) Washed with DMF (8 times for 20 sec. each)
5) Checked for free amino groups--Kaiser Test (must be positive)
6) The peptide resin was then gently shaken for 1 or 2 hrs with 8
equivalents of the desired FMOC-protected amino acid and
1-hydroxybenzotriazole and
benzotriazol-1-yl-oxy-tris(dimethyl-amino)phosphonium hexafluorophosphate
all dissolved in dry redistilled DMF containing 16 equivalents of
4-methylmorpholine.
7) Washed with DMF (6 times for 20 sec. each)
After step 7, an aliquot was taken for a ninhydrin test. If the test was
negative, one goes back to step 1 for coupling of the next amino acid. If
the test was positive or slightly positive, steps 6 and 7 should be
repeated.
The above scheme maybe used for coupling each of the amino acids of the
peptides described in this invention. N.alpha.-protection with FMOC may
also be used with each of the remaining amino acids throughout the
synthesis.
Radiolabeled peptides may be prepared by incorporation of a tritiated amino
acid using the above coupling protocol.
After the addition of the last amino acid, the N.alpha.-FMOC of the
N-terminal residue is removed by going back to steps 1-7 of the above
scheme. The peptide resin is washed with CH.sub.2 Cl.sub.2 and dried in
vacuo to give the crude protected peptide.
Procedure 3
Deprotection and Cleavage of the Peptides From the Resin
The protected peptide-resin was suspended in a 55% solution of
trifluoroacetic acid (TFA) in CH.sub.2 Cl.sub.2, containing 2.5%
ethanedithiol and 2.5% anisole. The mixture was flushed with N.sub.2 and
stirred for 1.5 hours at room temperature. The mixture was filtered and
the resin washed with CH.sub.2 Cl.sub.2. The resin was treated again with
20% TFA in CH.sub.2 Cl.sub.2 for 5 minutes at room temperature. The
mixture was filtered and the resin washed with 20% TFA in CH.sub.2
Cl.sub.2 and then washed with CH.sub.2 Cl.sub.2. The combined filtrates
were evaporated in vacuo below 35.degree. C. and the residue triturated
several times with dry dimethyl ether. The solid was dissolved in 10%
aqueous acetic acid and lyophilized to afford the crude product.
The peptides containing arg and cys residues are further deprotected by HF
treatment at 0.degree. C. for 1 hour in the presence of anisole and
dimethylsulfide. The peptides were extracted with 10% aqueous acetic acid,
washed with dimethyl ether and lyophilized to afford the crude peptides.
Procedure 4
Purification of Peptides
The crude peptides were purified by preparative HPLC on a Vydac column
(2.5.times.25 mm) of C.sub.18 or C.sub.4 reverse phase with a gradient of
the mobile phase. The effluent was monitored at 220 nm and subsequently by
analytical HPLC. Relevant fractions were pooled, evaporated and
lyophilized. The identity of the synthetic peptides was verified by
analytical reverse phase chromatography and by amino acid analysis.
Procedure 5
Cyclization of Peptides
A solution of potassium ferricyanide (0.01M, pH 7.0) was added slowly to a
dilute aqueous solution (0.5 mM) of the linear peptide at pH 7.0. After 24
hours at room temperature, the pH was lowered to 5.0 and the solution
treated with ion exchange resin (Bio-Rad Ag-3-X4a, Cl-form) for 30
minutes. The suspension was filtered and the filtrate lyophilized to give
the crude cyclic peptide. The peptide was purified by preparative reverse
phase HPLC and characterized by amino acid analysis. Proof of cyclicity
was obtained by comparing the HPLC mobility of the cyclic peptide with the
starting linear peptide by reducing an aliquot of the cyclic peptide back
to the linear peptide and also by observing the disappearance of free
sulfhydryl groups (Ellman's Test) after the cyclization.
Procedure 6
Conjugation of Peptides to Bovine Serum Albumin or Keyhole Limpet
Hemocyanin
Peptides were conjugated to BSA or KLH previously derivatized with either
sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Sulfo-SMPB) or
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC).
An aqueous solution of sulfo-SMPB or sulfo-SMCC (Pierce Chemicals) was
added to a solution of BSA or KLH in 0.02 M sodium phosphate buffer (pH
7.0). The mixture was shaken at room temperature for 45 minutes and the
activated carrier immediately applied to a Sephadex G-25 column
equilibrated with 0.1M sodium phosphate buffer (pH 6.0) at 4.degree. C.
The fractions of the first peak absorbance (280 nm) corresponding to
activated carrier were combined in a round bottom flask to which was added
a solution of peptide in 0.05 M sodium phosphate buffer (pH 6.2). The
mixture was thoroughly flushed with N.sub.2 and incubated overnight at
room temperature. The coupling efficiency was monitored using .sup.3
H-labeled peptide and by amino acid analysis of the conjugate.
Procedure 7
Detection of Antibodies to HIV by an Enzyme Linked Immunosorbent Assay
(ELISA)
Each well of the microtiter plate is saturated with 100 .mu.l of a solution
containing a peptide or mixture of peptides (5 .mu.g/ml) and left
overnight. The wells are emptied and washed twice with a washing buffer
(Tris, 0.043M; NaCl, 0.5M; thimerosal, 0.01% w/v; Tween 20, 0.05% v/v; pH
7.4). The wells are then saturated with 0.35 ml of washing buffer for 1
hr. at 37.degree. C. and washed once with the same buffer. Serum samples
to be analyzed are diluted with specimen buffer (washing buffer plus
casein, 0.05% w/v). The wells are rinsed with washing buffer prior to the
addition of the diluted serum sample (0.1 ml). These are left to incubate
for 1 hr. at room temperature. The wells are then emptied, washed twice
rapidly and then once for two minutes with washing buffer. The conjugate
solution (affinity purified goat antibody to human IgG peroxidase labeled,
0.5 mg in 5 ml 50% glycerol) diluted with 1% w/v bovine serum albumin in
washing buffer is added to each well (0.1 ml) and incubated for 1 hr. at
room temperature. The wells are then emptied and washed twice rapidly with
washing buffer and then five times in which the buffer was in contact with
the well 2 minutes per washing. The substrate solution
(3,3',5,5'-tetramethylbenzidine, 8 mg per ml of DMSO) is diluted with 100
volumes 0.1M citrate-acetate buffer, pH 5.6 containing 0.1% v/v of 30%
H.sub.2 O.sub.2 and added to each well (0.1 ml per well). After 10 minutes
the contents of each well is treated with 0.1 ml 2N H.sub.2 SO.sub.4 and
the optical density read at 450 nm. All determinations are done in
duplicate.
Procedure 8
Preparation of Peptide Cocktails
Peptide cocktails were prepared by mixing together equal volumes of two
peptide solutions each at 10 ug/ml. One cocktail used peptides BCH-87c and
BCH-202c and the other cocktail was a mixture of BCH-87ck and BCH-202ck.
Each cocktail was used to coat two series of plates as described earlier.
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